cDNA cloning of inositol monophosphatase

ABSTRACT

The present invention relates to the cloning of novel cDNA sequences encoding human and rat inositol monophosphatase (IMP); to the preparation of IMP enzyme by incorporation of the cDNAs into an expression vector and the expression thereof in recombinant host cells; and to the use of the enzyme thereby obtained in designing and developing medicaments which are inhibitors of human or rat IMP.

The present invention relates to cDNA encoding inositol monophosphatase(IMP) which is isolated from brain cells. As used herein, theabbreviation IMP refers to an enzyme which can specifically liberateinositol (Ins) from the naturally occurring substrates Ins(1)P, Ins(3)Pand Ins(4)P. IMP is also capable of hydrolyzing certainnon-inositol-containing substrates including but not limited to thosedisclosed in Hallcher and Sherman, (1980), J. Biol. Chem., 224, pp.10896-10901; Takimoto et al., (1985), J. Biochem, (Tokyo), 98, pp.363-370; and Gee et al., (1988), Biochem. J., 249, pp. 883-889.

The amino acid and cDNA sequence of bovine IMP is known Diehl et al.,(1989), J. Biol. Chem., 265, pp. 5946-5949!. Mammalian cells capable ofproducing IMP include, but are not limited to, brain tissue cells.Transformed mammalian cell lines which may produce IMP include, but arenot limited to, brain derived cell lines such as those available fromthe American Type Culture Collection listed in the Catalogue of Celllines & Hybridomas, 7th Edition, 1992. The preferred cells for thepresent invention include normal human brain-derived tissue cells.

Other cells and cell lines may also be suitable for use in isolating IMPcDNA. Selection of suitable cells may be done by screening for IMPactivity in cell extracts or conditioned medium. Methods for detectingIMP activity are well known in the art Ragan et al., (1988), Biochem.J., 249, pp. 143-148!, and measure the liberation of ¹⁴ C-labelledinositol from a substrate. Cells which possess IMP activity in thisassay may be suitable for the isolation of IMP cDNA.

Any of a variety of procedures may be used to molecularly clone humanIMP cDNA. These methods include, but are not limited to, directfunctional expression of the human IMP cDNA following the constructionof a human IMP containing cDNA library in an appropriate expressionvector system. Another method is to screen a human IMP-containing cDNAlibrary constructed in a bacteriophage or plasmid shuttle vector with alabelled oligonucleotide probe designed from the amino acid sequence ofthe purified IMP protein or from the DNA sequence of bovine IMP cDNA.The preferred method consists of screening a human IMP-containing cDNAlibrary constructed in a bacteriophage or plasmid shuttle vector with a³² P-labelled cDNA oligonucleotide-primed fragment of the bovine IMPcDNA Diehl et al., (1989), J. Biol. Chem., 265, pp. 5946-5949!. Thepreferred cDNA library is a commercially available human hippocampalcDNA library in lambdaZAP (Stratagene).

It is readily apparent to those skilled in the art that other types oflibraries, as well as libraries constructed from other cells or celltypes, may be useful for isolating IMP-encoding DNA. Other types oflibraries include, but are not limited to, cDNA libraries derived fromother tissues, cells or cell lines other than human hippocampal cells,and genomic DNA libraries.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have IMPactivity. The selection of cells or cell lines for use in preparing acDNA library to isolate IMP cDNA may be done by first measuringcell-associated IMP activity using the ¹⁴ C-labelled inositol substratecleavage assay described above Ragan et al., supra!.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found, for example, in Maniatis, T., Fritsch, E. F., Sambrook,J., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press,New York, 2nd edition, 1989).

It is also readily apparent to those skilled in the art that DNAencoding IMP may also be isolated from a suitable genomic DNA library.

Construction of genomic DNA libraries can be performed by standardtechniques well known in the art. Well known genomic DNA libraryconstruction techiques can be found in Maniatis et al., supra.

Using the preferred method, cDNA clones encoding human IMP were isolatedby cDNA library screening. ³² P-radiolabelled oligonucleotide-primedfragments of the bovine IMP cDNA served as probes for the isolation offull length human IMP cDNA from a commercially available lambdaZAP cDNAlibrary (Stratagene) derived from human hippocampal cells.

Three positively hybridising phage, designated lambdaHIMP1-3, weredetected using the bovine IMP cDNA-derived probe. These cDNA clonescontained an insert of about 2 kilobases (kb) in length and had a singleopen reading frame of about 277 amino acids. Following in vivo excisionaccording to the standard protocol as supplied by the manufacturer(Stratagene), three plasmids were obtained, designated pHIMP1-3respectively. These positive cDNA clones were bi-directionally sequencedby the dideoxy chain termination method (Sanger et al., P.N.A.S. USA,74, 5463, 1977).

The sequence (SEQ. ID NO. 1) for the full-length cDNA encoding humanbrain IMP is shown in Table 1, and was derived from clone pHIMP3. Thededuced amino acid sequence (SEQ. ID NO. 2) of human IMP from the clonedcDNA is shown alongside the cDNA sequence in Table 1. Inspection of thecDNA sequence reveals the presence of a single, large open reading frameof 277 amino acids. A comparison of the bovine IMP amino acid sequencewith the human IMP amino acid sequence shows about 85% sequenceidentity.

                                      TABLE 1                                     __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                    __________________________________________________________________________

In addition to the isolation and cloning of human brain IMP cDNA, ratbrain IMP cDNA was also isolated and cloned from a cDNA library. Acommercially available rat brain cDNA library (Clontech) construoted inlambdaGT11 was screened using ³² P-radiolabelled oligonucleotide-primedfragments of the bovine IMP cDNA as probes. Plaques were plated and twopositive clones were identified. Both clones contained DNA inserts ofapproximately 2.1 kb which were sequenced. The DNA inserts wereidentical and contained an open reading frame of 270 amino acidsbeginning from amino acid residue 8 of the human brain IMP DNA sequence(cf. Table 1). The sequence of this cDNA insert, together with thededuced amino acid sequence (SEQ. ID NO. 4) corresponding thereto, isshown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                     ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                     ##STR25##                                                                     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                     ##STR30##                                                                     ##STR31##                                                                     ##STR32##                                                                     ##STR33##                                                                     ##STR34##                                                                    __________________________________________________________________________

IMP in substantially pure form derived from natural sources is found tobe an association of two IMP polypeptides apparently encoded by a singlemRNA Gee et al., Biochem. J., 1988, 549, 883-889!. The two IMPpolypeptides are found to have an apparent molecular weight of about 30kDa.

The cloned IMP cDNA obtained through the methods described above may berecombinantly expressed by molecular cloning into an expression vectorcontaining a suitable promoter and other appropriate transcriptionregulatory elements, and then transferring the expression vector intoprokaryotic or eukaryotic host cells to produce recombinant IMP.Molecular cloning of cDNAs into an expression vector can be achieved bya variety of standard techniques; in the present context, polymerasechain reaction (PCR) methodology Saiki et al., Science, 1988, 239,487-491! is preferably employed. Alternative conventional techniques forsuch manipulations are fully described in Maniatis et al., supra, andare well known in the art.

Expression vectors are defined herein as DNA sequences that are requiredfor the transcription of cloned copies of genes and the translation oftheir mRNAs in an appropriate host. Such vectors can be used to expresseukaryotic genes in a variety of hosts such as bacteria, bluegreenalgae, plant cells, insect cells and animal cells.

Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector should contain: an origin of replicationfor autonomous replication in host cells, selectable markers, a limitednumber of useful restriction enzyme sites, a potential for high copynumber, and active promoters. A promoter is defined as a DNA sequencethat directs RNA polymerase to bind to DNA and initiate RNA synthesis. Astrong promoter is one which causes mRNAs to be initiated at highfrequency. Expression vectors may include, but are not limited to,cloning vectors, modified cloning vectors, specifically designedplasmids or viruses.

A variety of mammalian expression vectors may be used to expressrecombinant IMP in mammalian cells. Commercially available mammalianexpression vectors which may be suitable for recombinant IMP expressioninclude, but are not limited to, pRSET5a Schoepfer et al., FEBS Lett.,1990, 257, 393-399!, pMClneo (Stratagene), pXT1 (Stratagene), pSG5(Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2) (ATCC 37110),pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and gZD35 (ATCC37565).

DNA encoding IMP cloned into an expression vector may then betransferred to a recombinant host cell for expression. Recombinant hostcells may be prokaryotic or eukaryotic, including but not limited tobacteria, yeast, mammalian cells (including but not limited to celllines of human, bovine, porcine, monkey and rodent origin), and insectcells (including but not limited to drosophila derived cell lines). Apreferred bacterial cell line is BL21-DE3 Schoepfer et al., FEBS Lett.,1990, 257, 393-399!. Cell lines derived from mammalian species which maybe suitable and which are commercially available include, but are notlimited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658),HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5(ATCC CCL 171).

It will be appreciated that, if the cDNA is to be inserted into theexpression vector pRSET5a, then this expression vector must be used inconjunction with a recombinant host containing the T7 polymerase gene.One such host is the bacterial strain BL21-DE3. Thus, a preferred methodin the present case is to insert the cDNA into the expression vectorpRSET5a and express this vector in the bacterial strain BL21-DE3.

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transformation,transfection, protoplast fusion, and electroporation. The expressionvector-containing cells are clonally propagated and individuallyanalyzed to determine whether they produce IMP protein. Identificationof IMP expressing host cell clones may be done by several means,including but not limited to immunological reactivity with anti-IMPantibodies, and the presence of host cell-associated IMP activity.

Expression of IMP DNA may also be performed using in vitro producedsynthetic mRNA. Synthetic mRNA can be efficiently translated in variouscell-free systems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell basedsystems, including but not limited to microinjection into frog oocytes.

To determine the IMP cDNA sequence(s) that yield(s) optimal levels ofenzymatic activity and/or IMP protein, IMP cDNA molecules including butnot limited to the following can be constructed: the full-length openreading frame of the IMP cDNA (base 1 to base 834; Table 1) andconstructs containing portions of the cDNA encoding enzymatically activeprotein. All constructs can be designed to contain none, all or portionsof the 5' and 3' untranslated region of IMP cDNA. A particular constructis one which is substantially free of the 5' untranslated region of IMPcDNA. IMP activity and levels of protein expression can be determinedfollowing the introduction, both singly and in combination, of theseconstructs into appropriate host cells. Following determination of theIMP cDNA cassette which yields optimal expression, this IMP cDNAconstruct may be transferred to a variety of expression host cells,including but not limited to mammalian cells, baculovirus-infectedinsect cells, E. coli, and the yeast S. cerevisiae.

Expression of IMP in a recombinant host cell affords IMP protein inactive form, capable of enzymatically liberating inositol from naturallyoccurring IMP substrates. Several IMP purification procedures areavailable and suitable for use. Recombinant IMP may be purified fromcell lysates and extracts, or from conditioned culture medium, byvarious combinations of, or individual application of, saltfractionation, ion exchange chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography. The preferred method ision-exchange chromatography utilizing a column of HR5/5 Mono Q matrix(LKB Pharmacia).

In addition, recombinant IMP can be separated from other cellularproteins by use of an immuno-affinity column made with monoclonal orpolyclonal antibodies specific for IMP, or polypeptide fragments of IMP.The preparation and purification of monoclonal or polyclonal antibodiesspecific for IMP or polypeptide fragments thereof can be accomplished byconventional techniques well known in the art. Typical proceduresinclude those described, for example, by Maniatis et al. in MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, 2nd edition,1989, Chapter 18.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes IMP, preferably human IMP, so as to prevent translation ofthe mRNA molecule. The antisense oligonucleotide may have a sequencecapable of binding specifically with any sequences of the cDNA moleculewhose sequence is shown in Table 1 or the cDNA molecule whose sequenceis shown in Table 2. A particular example of an antisenseoligonucleotide is an antisense oligonucleotide comprising chemicalanalogues of nucleotides.

This invention provides a transgenic nonhuman mammal expressing DNAencoding IMP, preferably human IMP. This invention also provides atransgenic nonhuman mammal expressing DNA encoding IMP, preferably humanIMP, so mutated as to be incapable of normal receptor activity, and notexpressing native IMP. This invention further provides a transgenicnonhuman mammal whose genome comprises antisense DNA complementary toDNA encoding IMP, preferably human IMP, so placed as to be transcribedinto antisense mRNA which is complementary to mRNA encoding IMP andwhich hybridizes to mRNA encoding IMP thereby reducing its translation.The DNA may additionally comprise an inducible promoter or additionallycomprise tissue specific regulatory elements, so that expression can beinduced, or restricted to specific cell types. Examples of DNA are DNAor cDNA molecules having a coding sequence substantially the same as thecoding sequence shown in Table 1, or the coding sequence shown in Table2. An example of a transgenic animal is a transgenic mouse. Examples oftissue specificity-determining regions are the metallothionein promotor(Low et al., Science, 1986, 231, 1002-1004) and the L7 promotor(Oberdick et al., Science, 1990, 248, 223-226).

The cloned IMP enzyme in accordance with the invention, when isolated,may be utilised to generate structural data, in particular X-raycrystallographic data. Knowledge of, for example, the X-ray crystalstructure of the IMP enzyme, especially human IMP, may facilitate indesigning drugs which are inhibitors of this enzyme. The presentinvention accordingly provides the use of the cloned IMP enzyme inscreening for and designing medicaments which are inhibitors of IMP.

The following non-limiting Examples illustrate the present invention.

EXAMPLE 1 Cloning of Human and Rat Brain Inositol Monophosphatase

A human hippocampal cDNA library constructed in lambdaZAP (Stratagene)was screened using a ³² P-radiolabelled oligonucleotide-primed fragmentof bovine inositol monophosphatase cDNA as a probe Diehl et al., (1990),J. Biol. Chem., 265, pp. 5946-5949!. Phage (100,000) were plated andthree independent cDNA clones were isolated. All three contained aninsert of approximately 2 kb which were subsequently shown to haveidentical sequences. One of these clones was characterized further andfound to contain a 277 amino acid open reading frame (Table 1). Thepredicted amino acid sequence (Table 1) is very similar to that of thebovine enzyme and has an estimated subunit M_(r) of approximately30,000, as does the bovine enzyme Gee et al., (1988), Biochem. J., 249,pp. 883-889!.

Additionally, a rat brain cDNA library constructed in lambdaGT11(Clontech) was screened using ³² P-radiolabelled oligonucleotide-primedfragments of bovine inositol monophosphatase cDNA as a probe asdescribed above. Phage were plated and two independent cDNA clones wereisolated. Each contained an insert of approximately 2.1 kb which weresubsequently shown to have identical sequences. One clone wascharacterized further and found to contain a 270 amino acid open readingframe beginning with amino acid 8 of the human brain IMP cDNA. The ratbrain IMP cDNA sequence, together with the deduced amino acid sequencecorresponding thereto, is shown in Table 2.

EXAMPLE 2 Expression of Recombinant Human Inositol Monophosphatase inBacteria

The T7 polymerase bacterial expression system (pRSET5a) was used asdescribed previously Diehl et al., supra!. The coding region of thehuman inositol monophosphatase cDNA was reconstructed to contain an NdeIsite at the start codon and a PstI site just downstream of the stopcodon using PCR methodology Saiki et al., (1988), Science, 239, pp.487-491!. Oligonucleotides (SEQ. ID NO. 5)5'-AATATTTTCAGCATATGGCTGATCCTTG-3' and (SEQ. ID NO. 6)5'-ATGACTATGAGCTGCAGTAATTAATCTTC-3' were synthesized on an AppliedBiosystems 380B instrument. Inositol monophosphatase cDNA (100 ng) inpBluescript II SK (Stratagene Ltd., U.K.) was subjected to PCR understandard conditions Saiki, et al., supra!, denaturation at 94° C. for 2minutes, annealing at 55° C. for 2 minutes and polymerisation at 72° C.for 6 minutes. Twenty cycles were performed with the last polymerisationstep lasting 12 minutes. The NdeI/PstI-digested PCR product was clonedinto NdeI-PstI-digested pRSET5a and transformed into Escherichia colistrain DH5α competent cells. Positive clones were identified byrestriction analysis and DNA sequencing. For subsequent expressionstudies, the expression vector was transformed into competent BL21-DE3cells. Bacterial cells containing the expression vector were induced byisopropylthio-β-D-galactoside and analysed by both SDS/PAGE and enzymeassay.

Recombinant bacteria expressed a prominent polypeptide of about M_(r)30,000 which was absent from bacteria not containing the expressionvector. Bacterial lysates contained significant amounts of enzymeactivity, which was further purified by heat treatment, centrifugationand ion-exchange chromatography (Example 3).

EXAMPLE 3 Purification of Recombinant Human Inositol Monophosphatase

E. coli bacteria (strain BL21-DE3) containing the IMP expression vector(Example 2) were grown and induced by isopropylthio-β-D-galactoside, andanalysed by SDS/PAGE and enzyme assay as described previously Ragan etal., supra!. After induction, cells were pelleted and frozen untilrequired for purification. THe bacterial pellets (1-2 g/liter offermentation mixture) were thawed, resuspended in 10 volumes of 20mM-TRIS/HCl/1 mM-EGTA buffer, pH 7.8 (buffer A) and sonicated on ice(3×1 min). The homogenate was then centrifuged at 100,000 xg for 20minutes and the resultant supernatant was loaded, at a flow rate of 1ml/min, on an HR5/5 Mono Q column (LKB Pharmacia), previouslyequilibrated with buffer A. The column was eluted at 1 ml/min with agradient of 0-300 mM NaCl in buffer A, and 1 ml fractions werecollected. Portions (5 μl) of each fraction were subjected to SDS/PAGEon 12% gels according to the method of Laemmli, (1970), Nature, 227, pp.680-685. Gels were stained with Coomassie Blue and the appropriateinositol monophosphatase-containing fractions were pooled for subsequentanalysis.

SDS/PAGE of a typical purification shows a single band of about M_(r)30,000. Table 3 shows a 5.4-fold purification of the enzyme,demonstrating that recombinant inositol monophosphatase constitutednearly 20% of the original bacterial protein. This level of expressionenables the isolation of large amounts of pure enzyme for X-raycrystallography and other studies. The biochemical properties of thehuman enzyme were further examined.

                  TABLE 3                                                         ______________________________________                                                   Total    Total     Specific                                                   activity protein   activity                                                                              Yield                                   Step       (m units)                                                                              (mg)      (m units/mg)                                                                          (%)                                     ______________________________________                                        Bacterial  751,100  214       3500    100                                     homogenate                                                                    Bacterial  481,000  90        5300    64                                      supernatant                                                                   Heat-treated                                                                             264,000  22        12,000  35                                      supernatant                                                                   Mono Q     177,000  9         19,700  24                                      ______________________________________                                    

EXAMPLE 4 Kinetic Properties of Recombinant Human InositolMonophosphatase

In agreement with previous work on the rat and bovine enzyme Takimoto etal., (1985), J. Biochem., 18, pp. 363-370; Gee et al., supra! we foundthat human brain IMP activity was totally dependent on Mg²⁺. Understandard assay conditions, the apparent affinity of the human enzyme forMg²⁺ was approximately 2-fold greater than that of the bovine enzyme,and the human enzyme was also more sensitive to inhibition by high Mg²⁺concentrations. In the absence of KCl, activation by Mg²⁺ occurred atapproximately 2-fold lower concentrations, but maximum enzyme activitywas independent of KCl. As is shown in Table 4, the recombinant humanbrain enzyme has similar kinetic properties to the native humanplatelet-derived enzyme as well as both the native and recombinantbovine enzymes Gee et al., supra; Diehl et al., supra!. The K_(m) forDL-Ins(1)P was lower for the human enzyme, suggesting that it may have ahigher affinity for substrate than the bovine enzyme. These data alsosuggest that there is no significant post-translational modification ofthe native human enzyme that might change its behaviour compared withthe recombinant enzyme.

                  TABLE 4                                                         ______________________________________                                        Kinetic properties of Inositol monophosphatases                                            K.sub.m  for  V.sub.max  (μmol/min                            Enzyme       DL-Ins(1)P (mM)                                                                             per mg of protein)                                 ______________________________________                                        Human        0.075 ± 0.003                                                                            36.8 ± 1                                        recombinant                                                                   Human        0.108 ± 0.003                                                                            ND                                                 platelet                                                                      Bovine       0.12 ± 0.007                                                                             ND                                                 recombinant                                                                   Bovine brain 0.16 ± 0.02                                                                              13.3 ± 0.9                                      ______________________________________                                    

Initial rates determined with several substrate concentrations werefitted to a Michaelis-Menten expression by non-linear least-squaresregression analysis. Values for K_(m) and V_(max) were determined in thepresent study and are given as means ±S.E.M. (n=3). Bovine brain valuesare as reported in Gee et al., supra, but confirmed here.

EXAMPLE 5 Inhibition of Recombinant Human Inositol Monophosphatase

P_(i) inhibited recombinant human IMP competitively with a K_(i) valueof 0.14 mM (cf. bovine enzyme, 0.5 mM), and Li⁺ was an uncompetitiveinhibitor with an apparent K_(i) value of 0.3 mM (cf. bovine enzyme 0.26mM). The competitive inhibitor1S-phosphoryloxy-2R,4S-dihydroxycyclohexane Baker et al., (1990), J.Chem. Soc. Chem. Comm., pp. 462-464! inhibited with an apparent K_(i)value of 2.7 μM (cf. bovine enzyme, 1.1 μM). All comparisons are fromdata obtained in parallel experiments carried out in this study.Overall, these data confirm that the human enzyme is similar to, but notidentical with, the bovine enzyme. The availability of multiple proteinsequences and recombinant human enzyme will assist future studies usingsite-directed mutagenesis and chemical-modification techniques tocharacterize important residues for the structure and function of thisenzyme.

IMP Enzyme Assays

Enzyme activity was determined by measuring the release of ¹⁴ C!inositolfrom DL-Ins(1)P containing L- U-¹⁴ C!Ins(1)P as label as describedpreviously Gumber et al., (1989), Plant Physiol., 76, pp. 40-44!. Oneunit of enzyme activity represents 1 μmol of substrate hydrolysed/min,at 37° C. Protein concentrations were determined by the method ofBradford Bradford, M., (1976), Anal. Biochem., 72, pp. 248-252!. Kineticanalyses were performed as described previously Gee et al., supra!.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 897 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 37..871                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      CTCCGACTCAAGATATTTGTCAAATATTTTCAGAAGATGGCTGATCCTTGGCAG54                      MetAlaAspProTrpGln                                                            15                                                                            GAATGCATGGATTATGCAGTAACTCTAGCAAGACAAGCTGGAGAGGTA102                           GluCysMetAspTyrAlaValThrLeuAlaArgGlnAlaGlyGluVal                              101520                                                                        GTTTGTGAAGCTATAAAAAATGAAATGAATGTTATGCTGAAAAGTTCT150                           ValCysGluAlaIleLysAsnGluMetAsnValMetLeuLysSerSer                              253035                                                                        CCAGTTGATTTGGTAACTGCTACGGACCAAAAAGTTGAAAAAATGCTT198                           ProValAspLeuValThrAlaThrAspGlnLysValGluLysMetLeu                              404550                                                                        ATCTCTTCCATAAAGGAAAAGTATCCATCTCACAGTTTCATTGGTGAA246                           IleSerSerIleLysGluLysTyrProSerHisSerPheIleGlyGlu                              55606570                                                                      GAATCTGTGGCAGCTGGGGAAAAAAGTATCTTAACCGACAACCCCACA294                           GluSerValAlaAlaGlyGluLysSerIleLeuThrAspAsnProThr                              758085                                                                        TGGATCATTGACCCTATTGATGGAACAACTAACTTTGTACATAGATTT342                           TrpIleIleAspProIleAspGlyThrThrAsnPheValHisArgPhe                              9095100                                                                       CCTTTTGTAGCTGTTTCAATTGGCTTTGCTGTAAATAAAAAGATAGAA390                           ProPheValAlaValSerIleGlyPheAlaValAsnLysLysIleGlu                              105110115                                                                     TTTGGAGTTGTGTACAGTTGTGTGGAAGGCAAGATGTACACTGCCAGA438                           PheGlyValValTyrSerCysValGluGlyLysMetTyrThrAlaArg                              120125130                                                                     AAAGGAAAAGGGGCCTTTTGTAATGGTCAAAAACTACAAGTTTCACAA486                           LysGlyLysGlyAlaPheCysAsnGlyGlnLysLeuGlnValSerGln                              135140145150                                                                  CAAGAAGATATTACCAAATCTCTCTTGGTGACTGAGTTGGGCTCTTCT534                           GlnGluAspIleThrLysSerLeuLeuValThrGluLeuGlySerSer                              155160165                                                                     AGAACACCAGAGACTGTGAGAATGGTTCTTTCTAATATGGAAAAGCTT582                           ArgThrProGluThrValArgMetValLeuSerAsnMetGluLysLeu                              170175180                                                                     TTTTGCATTCCTGTTCATGGGATCCGGAGTGTTGGAACAGCAGCTGTT630                           PheCysIleProValHisGlyIleArgSerValGlyThrAlaAlaVal                              185190195                                                                     AATATGTGCCTTGTGGCAACTGGCGGAGCAGATGCATATTATGAAATG678                           AsnMetCysLeuValAlaThrGlyGlyAlaAspAlaTyrTyrGluMet                              200205210                                                                     GGAATTCACTGCTGGGATGTTGCAGGAGCTGGCATTATTGTTACTGAA726                           GlyIleHisCysTrpAspValAlaGlyAlaGlyIleIleValThrGlu                              215220225230                                                                  GCTGGTGGCGTGCTAATGGATGTTACAGGTGGACCATTTGATTTGATG774                           AlaGlyGlyValLeuMetAspValThrGlyGlyProPheAspLeuMet                              235240245                                                                     TCACGAAGAGTAATTGCTGCAAATAATAGAATATTAGCAGAAAGGATA822                           SerArgArgValIleAlaAlaAsnAsnArgIleLeuAlaGluArgIle                              250255260                                                                     GCTAAAGAAATTCAGGTTATACCTTTGCAACGAGACGACGAAGATTAAT871                          AlaLysGluIleGlnValIleProLeuGlnArgAspAspGluAsp*                                265270275                                                                     TAAGGCAGCTCATAGTCATCCAGTTG897                                                 (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 277 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      MetAlaAspProTrpGlnGluCysMetAspTyrAlaValThrLeuAla                              151015                                                                        ArgGlnAlaGlyGluValValCysGluAlaIleLysAsnGluMetAsn                              202530                                                                        ValMetLeuLysSerSerProValAspLeuValThrAlaThrAspGln                              354045                                                                        LysValGluLysMetLeuIleSerSerIleLysGluLysTyrProSer                              505560                                                                        HisSerPheIleGlyGluGluSerValAlaAlaGlyGluLysSerIle                              65707580                                                                      LeuThrAspAsnProThrTrpIleIleAspProIleAspGlyThrThr                              859095                                                                        AsnPheValHisArgPheProPheValAlaValSerIleGlyPheAla                              100105110                                                                     ValAsnLysLysIleGluPheGlyValValTyrSerCysValGluGly                              115120125                                                                     LysMetTyrThrAlaArgLysGlyLysGlyAlaPheCysAsnGlyGln                              130135140                                                                     LysLeuGlnValSerGlnGlnGluAspIleThrLysSerLeuLeuVal                              145150155160                                                                  ThrGluLeuGlySerSerArgThrProGluThrValArgMetValLeu                              165170175                                                                     SerAsnMetGluLysLeuPheCysIleProValHisGlyIleArgSer                              180185190                                                                     ValGlyThrAlaAlaValAsnMetCysLeuValAlaThrGlyGlyAla                              195200205                                                                     AspAlaTyrTyrGluMetGlyIleHisCysTrpAspValAlaGlyAla                              210215220                                                                     GlyIleIleValThrGluAlaGlyGlyValLeuMetAspValThrGly                              225230235240                                                                  GlyProPheAspLeuMetSerArgArgValIleAlaAlaAsnAsnArg                              245250255                                                                     IleLeuAlaGluArgIleAlaLysGluIleGlnValIleProLeuGln                              260265270                                                                     ArgAspAspGluAsp                                                               275                                                                           (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 909 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..813                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      TGCATGGATTATGCAGTGATCCTCGCAAGACAAGCTGGAGAGATGATT48                            CysMetAspTyrAlaValIleLeuAlaArgGlnAlaGlyGluMetIle                              151015                                                                        CGAGTTGCTCTAAAAAATAAGATGGATGTCATGATTAAAAGTTCTCCA96                            ArgValAlaLeuLysAsnLysMetAspValMetIleLysSerSerPro                              202530                                                                        GCCGACTTGGTAACAGTTACTGACCAAAAAGTTGAAAAAATGCTTATG144                           AlaAspLeuValThrValThrAspGlnLysValGluLysMetLeuMet                              354045                                                                        TCTTCTATAAAGGAAAAATACCCATATCACAGTTTCATTGGTGAAGAA192                           SerSerIleLysGluLysTyrProTyrHisSerPheIleGlyGluGlu                              505560                                                                        TCTGTGGCAGCCGGGGAAAAGACAGTCTTCACAGAGCAGCCCACGTGG240                           SerValAlaAlaGlyGluLysThrValPheThrGluGlnProThrTrp                              65707580                                                                      ATCATTGATCCCATTGACGGGACGACCAACTTTGTGCACCGGTTTCCC288                           IleIleAspProIleAspGlyThrThrAsnPheValHisArgPhePro                              859095                                                                        TTTGTAGCTGTTTCGATTGGCTTCGTTGTAAATAAAGAGATGGAGTTT336                           PheValAlaValSerIleGlyPheValValAsnLysGluMetGluPhe                              100105110                                                                     GGAGTTGTATACAGCTGTGTGGAAGATAAGATGTATACGGGCAGGAAA384                           GlyValValTyrSerCysValGluAspLysMetTyrThrGlyArgLys                              115120125                                                                     GGAAAAGGCGCCTTTTGTAACGGTCAGAAGCTTCGGGTCTCGCAGCAG432                           GlyLysGlyAlaPheCysAsnGlyGlnLysLeuArgValSerGlnGln                              130135140                                                                     GAAGACATTACCAAATCACTCTTGGTGACCGAGCTGGGATCGTCCAGA480                           GluAspIleThrLysSerLeuLeuValThrGluLeuGlySerSerArg                              145150155160                                                                  AAGCCGGAGACTTTGCGGATTGTTCTCTCCAACATGGAAAGGCTTTGC528                           LysProGluThrLeuArgIleValLeuSerAsnMetGluArgLeuCys                              165170175                                                                     TCCATTCCTATCCATGGAATCCGGAGTGTTGGGACAGCGGCTGTTAAT576                           SerIleProIleHisGlyIleArgSerValGlyThrAlaAlaValAsn                              180185190                                                                     ATGTGCCTTGTGGCAACGGGAGGAGCGGATGCCTATTACGAGATGGGG624                           MetCysLeuValAlaThrGlyGlyAlaAspAlaTyrTyrGluMetGly                              195200205                                                                     ATCCACTGCTGGGACATGGCTGGAGCTGGCATCATCGTCATAGAGGCT672                           IleHisCysTrpAspMetAlaGlyAlaGlyIleIleValIleGluAla                              210215220                                                                     GGCGGAGTGCTGCTGGATGTGACAGGTGGACCATTCGATTTGATGTCT720                           GlyGlyValLeuLeuAspValThrGlyGlyProPheAspLeuMetSer                              225230235240                                                                  CGGAGAATAATTGCTGCAAGTAATATAGCATTAGCAGAAAGAATAGCC768                           ArgArgIleIleAlaAlaSerAsnIleAlaLeuAlaGluArgIleAla                              245250255                                                                     AAAGAACTTGAGATAATACCTTTACAACGAGACGACGAAAGTTAGGCACGTA820                       LysGluLeuGluIleIleProLeuGlnArgAspAspGluSer                                    260265270                                                                     GAACCGCATCCAGCTCCGTCACACCTGCTCTCCCTGGGATGTTTAAAGATGTATGATGTC880               ACTGATTTAAATTTAACTTTGCAGTCCTG909                                              (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 270 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      CysMetAspTyrAlaValIleLeuAlaArgGlnAlaGlyGluMetIle                              151015                                                                        ArgValAlaLeuLysAsnLysMetAspValMetIleLysSerSerPro                              202530                                                                        AlaAspLeuValThrValThrAspGlnLysValGluLysMetLeuMet                              354045                                                                        SerSerIleLysGluLysTyrProTyrHisSerPheIleGlyGluGlu                              505560                                                                        SerValAlaAlaGlyGluLysThrValPheThrGluGlnProThrTrp                              65707580                                                                      IleIleAspProIleAspGlyThrThrAsnPheValHisArgPhePro                              859095                                                                        PheValAlaValSerIleGlyPheValValAsnLysGluMetGluPhe                              100105110                                                                     GlyValValTyrSerCysValGluAspLysMetTyrThrGlyArgLys                              115120125                                                                     GlyLysGlyAlaPheCysAsnGlyGlnLysLeuArgValSerGlnGln                              130135140                                                                     GluAspIleThrLysSerLeuLeuValThrGluLeuGlySerSerArg                              145150155160                                                                  LysProGluThrLeuArgIleValLeuSerAsnMetGluArgLeuCys                              165170175                                                                     SerIleProIleHisGlyIleArgSerValGlyThrAlaAlaValAsn                              180185190                                                                     MetCysLeuValAlaThrGlyGlyAlaAspAlaTyrTyrGluMetGly                              195200205                                                                     IleHisCysTrpAspMetAlaGlyAlaGlyIleIleValIleGluAla                              210215220                                                                     GlyGlyValLeuLeuAspValThrGlyGlyProPheAspLeuMetSer                              225230235240                                                                  ArgArgIleIleAlaAlaSerAsnIleAlaLeuAlaGluArgIleAla                              245250255                                                                     LysGluLeuGluIleIleProLeuGlnArgAspAspGluSer                                    260265270                                                                     (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      AATATTTTCAGCATATGGCTGATCCTTG28                                                (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      ATGACTATGAGCTGCAGTAATTAATCTTC29                                               __________________________________________________________________________

We claim:
 1. A DNA molecule encoding the human inositol monophosphatase corresponding to the amino acid sequence depicted in SEQ. ID NO. 2 herein.
 2. A DNA molecule corresponding to the nucleotide sequence depicted in SEQ ID NO.
 1. 3. A recombinant expression vector comprising the nucleotide sequence as claimed in claim 1 together with additional sequences capable of directing the synthesis of inositol monophosphatase.
 4. A recombinant host cell containing the expression vector as claimed in claim
 3. 5. A host cell as claimed in claim 4 which consists of the bacterial cell line BL21-DE3 containing the recombinant expression vector pRSET5a.
 6. A process for preparing recombinant inositol monophosphatase which comprises inserting the nucleotide sequence as claimed in claim 1 into an expression vector, incorporating the vector into a suitable host cell, and growing the host cell under conditions suitable for the expression of inositol monophosphatase.
 7. A protein exhibiting human inositol monophosphatase activity and corresponding to the amino acid sequence depicted in SEQ. ID NO. 2 herein. 